Analytical measurement of serum 25-OH-vitamin D3, 25-OH-vitamin D2 and their C3-epimers by LC–MS/MS in infant and pediatric specimens

Abstract

ObjectivesTo develop a simple and sensitive LC–MS/MS procedure for quantification of serum 25-OH-vitamin D3 (25-OH-D3), 25-OH-vitamin D2 (25-OH-D2), and their C3-epimers. MethodsSerum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC–MS/MS. Commercially available calibrators and QC materials were employed. The ion-transition 401.2→365.2 was monitored for 25-OH-D3 and C3-epi-25-OH-D3, 407.2→371.3 for d6-25-OH-D3, 413.2→331.2 for 25-OH-D2 and C3-epi-25-OH-D2 and 419.2→337.1 for, d6-25-OH-D2. As a proof-of-principle, 25-OH-D3 and C3-epi-25-OH-D3 were quantified in 200 pediatric subjects (0–20years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer. ResultsThe assay provided an LLOQ of ≤2.8nmol/L for all 25-OH-D metabolites, with a linear response up to 400nmol/L. The CV was <10% for 25-OH-D2/3 and <15% for C3-epi-25-OH-D3. C3-epi-25-OH-D3 was quantified in all subjects, with higher concentrations observed in infants ≤1year of age (11.44nmol/L vs. 4.4nmol/L; p<0.001). Within the first year of life, 25-OH-D3 concentrations increased linearly, while C3-epi-25-OH-D3 concentrations remained constant. At 12months of age, C3-epi-25-OH-D3 concentration dropped by almost 50% (11.4nmol/L in infants ≤1year of age vs. 5.4nmol/L in infants 1–2years of age; p<0.001). Liquid vitamin D3 supplements did not contain appreciable amounts of C3-epi-D3. ConclusionsThe proposed LC–MS/MS procedure is suitable for quantifying 25-OH-D3 metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1year of age and drops at 12months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer.